Temporal and spatial control of gene expression in metazoan organisms defines cellular heterogeneity, cell type-specific phenotypic specialization, and cell lineage stability. This proposal pursues the description of mechanisms for the establishment of selective and heritable regulatory chromatin landscapes and gene expression programs in distinct mammalian cell lineages. Specifically, utilizing a combination of novel mammalian model systems and established models and techniques I aim to determine how cell lineage specifying transcription factors affect and collaborate with characteristic lineage specifi regulatory chromatin features, such as incorporation of the histone variant H3.3 and monomethylation of H3K4 (H3K4me1), in directing cellular differentiation. The transcription factor PU.1 directs myeloid cell differentiation together with C/EBP1 and AP1, and is clearly linked, spatiotemporally, to lineage specific enhancers decorated with H3K4me1 and other characteristic enhancer histone code modifications. Using biochemical approaches, I propose to investigate the histone modifying enzyme activity and cofactor identity contained within myeloid lineage specifying transcription factor complexes. Transcription factors may directly recruit chromatin machinery to lineage specific enhancers, or alternatively, these factors may be sequentially recruited in a chromatin template dependent manner and the relative contributions of these possible mechanisms for establishing and propagating lineage specific chromatin landscapes will be assessed. Additionally, using well- established models of hematopoietic cell differentiation and phenotypic analysis combined with novel mouse models, the functional requirements for H3.3 in cellular differentiation and lineage maintenance will be assessed in vivo. Additionally, I will employ genomic analytical techniques for characterization of discrete functional effects of H3.3 on associated chromatin characteristics such as accessibility, enhancer transcription, and post-translational modification of histones. These studies will address an outstanding question in the field of chromatin biology and epigenetics, namely, how lineage specifying transcription factors template lineage specific chromatin landscapes defining developmental potential, responsiveness to secondary factors, and heritable transcription programs. Increased knowledge of mechanisms of cell fate determination will enhance our understanding of how these epigenetic mechanisms impact human health, during development and in tumorogenesis. PUBLIC HEALTH RELEVANCE: Eukaryotic life is characterized by thousands of differentiated cell types which all derive their programming from a single genome. These studies aim to reveal mechanisms for regulation of chromatin characteristics that control the establishment and heritable maintenance of cell type-specific gene expression programs. Alterations in these pathways can result in developmental defects and tumorigenesis, and therefore, an improved understanding of how transcription factors and chromatin features together control cellular differentiation and identity will have an important impact on human health.